Kinase inhibitors are an effective class of targeted therapy for oncology. However, heterogeneous patient response and acquired resistance continues to be a significant clinical and economic burden. To overcome this problem drug developers are pursuing novel kinase inhibitors for existing kinase targets with new modes of inhibition and increased potency, as well as compounds for emerging kinase targets. There is an unmet need in pre-clinical development for assays that measure endogenous kinase activity in disease-relevant cellular models. The long-term goal of this project is to commercialize an assay platform for measuring cell-based endogenous kinase activity. The goal of this Phase I proposal is to further establish feasibility of an assay platform based on cell-permeable peptide substrates specific for a kinase of interest. The peptide amino acid sequences are designed using a recently published algorithm developed by Purdue University researchers called KINATEST-ID. The proof-of-concept demonstration shows that KINATEST-ID designs specific substrates for cytosolic tyrosine kinases based on stringently curated endogenous kinase substrate sequences. In this proposal, successful completion of the aims will result in the expansion of the platform to show feasibility for receptor tyrosine kinases (RTKs). In the first ai we will identify endogenous substrates for RTKs in disease-relevant cellular models that will be used to design specific substrates for RTKs of interest using KINATEST-ID. This will be accomplished using a novel method known as kinase assay linked to phosphoproteomics (KALIP), which identifies endogenous sequences for the target RTK. The identified sequences will then be used as input data for the KINATEST-ID algorithm to design candidate RTK substrate sequences. In the second aim, designed substrate sequences will be synthesized and empirically validated for kinase specificity, and assay performance will be analytically characterized in vitro. Finally, substrates that meet quantitative criteria for specificity and assy performance in the second aim will be modified and optimized for use in cell-based applications. Successful completion of the aims will result in a panel of prototype cell-based assays for measuring endogenous RTK activity, and a validated R&D pipeline for further expansion of the assay platform. Phase II of this project will focus on developing partnerships to bring this assay platform to the drug discovery market, expansion into additional kinases, and adapting the assay for use in patient derived samples, such as biopsies or circulating tumor cells.